Mangafodipir protects against hepatic ischemia-reperfusion injury in mice

Abstract

Introduction and aim: Mangafodipir is a contrast agent used in magnetic resonance imaging that concentrates in the liver and displays pleiotropic antioxidant properties. Since reactive oxygen species are involved in ischemia-reperfusion damages, we hypothesized that the use of mangafodipir could prevent liver lesions in a mouse model of hepatic ischemia reperfusion injury. Mangafodipir (MnDPDP) was compared to ischemic preconditioning and intermittent inflow occlusion for the prevention of hepatic ischemia-reperfusion injury in the mouse.

Methods: Mice were subjected to 70% hepatic ischemia (continuous ischemia) for 90 min. Thirty minutes before the ischemic period, either mangafodipir (10 mg/kg) or saline was injected intraperitoneally. Those experimental groups were compared with one group of mice preconditioned by 10 minutes' ischemia followed by 15 minutes' reperfusion, and one group with intermittent inflow occlusion. Hepatic ischemia-reperfusion injury was evaluated by measurement of serum levels of aspartate aminotransferase (ASAT) activity, histologic analysis of the livers, and determination of hepatocyte apoptosis (cytochrome c release, caspase 3 activity). The effect of mangafodipir on the survival rate of mice was studied in a model of total hepatic ischemia.

Results: Mangafodipir prevented experimental hepatic ischemia-reperfusion injuries in the mouse as indicated by a reduction in serum ASAT activity (P<0.01), in liver tissue damages, in markers of apoptosis (P<0.01), and by higher rates of survival in treated than in untreated animals (P<0.001). The level of protection by mangafodipir was similar to that observed following intermittent inflow occlusion and higher than after ischemic preconditioning.

Conclusions: Mangafodipir is a potential new preventive treatment for hepatic ischemia-reperfusion injury.

Figure 1. Mangafodipir and intermittent clamping prevent the increase in transaminase activities following reperfusion injury.

Serum levels of aspartate amino-transferase activities (ASAT) served as markers of hepatocyte injury. ASAT levels were measured after 24 hours of reperfusion. Bars represent means ± SEM, nine mice in each group.

Figure 2. Mangafodipir and intermittent clamping prevent histological lesions of the liver following reperfusion injury.

Hematoxylin-eosin-stained sections were used for histopathological evaluation of hepatic injuries. The sections were examined at 400-fold magnification. Representative livers from normal BALB/c mice (A), Sham-operated mice (B), ninety minutes' ischemia control group (C), Mangafodipir (MnDPDP)-treated group (D), IP-treated group (E) and IC-treated group (F).

Figure 3. Prevention of lipid peroxidation following ischemia-reperfusion of the liver using on mangafodipir and intermittent clamping.

The concentrations of 4-hydroxyalkenals (4-HNE) and malondialdehyde (MDA) were measured spectrophotometrically in whole liver homogenates. The level of lipid peroxidation was expressed as the amounts of 4-HNE + MDA per mg of proteins. Bars represent means ± SEM; nine mice in each group.

Figure 4. Prevention of glutathione depletion in the mitochondria (A) and in the cytoplasm (B) of hepatocytes following reperfusion injury.

The concentrations of glutathione in cytoplasm and mitochondria of mouse liver were measured spectrophotometrically in whole liver homogenates. The levels of reduced gluthatione were expressed as pmol per 10 µg of proteins. Bars represent means ± SEM, nine mice in each group. The O2°- depletion in the mitochondria (C) of HepG2 cells induced by mangafodipir (400 µM) was assessed in a kinetic experiment. Immunofluorescence microscopy of HepG2 cells stained with oxidation of dihydroethidium (DHE) and treated for 18 h or not with mangafodipir. (D–E) A decreased intensity of cytosol with mangafodipir in mangafodipir treated cells was observed.

Figure 5. Prevention of hepatocytes apoptosis following reperfusion injury.

The mitochondrial/cytosol cytochrome c ratio (A) and caspase-3 activities were measured in the livers of the various experimental groups and controls. Caspase-3 like activities were expressed as Units per mg of proteins. Bars represent means ± SEM, nine mice in each group.